Enhanced Utilization of Phosphonate and Phosphite by Klebsiella aerogenes

Klebsiella aerogenes ATCC 9621 was able to utilize phosphonates (P(n)), including aminoethylphosphonate, ethylphosphonate, methylphosphonate (MP(n)), and phosphonoacetate, and inorganic phosphite (P(t)) as sole sources of phosphorus (P). The products of the phn gene cluster were absolutely required for P(n) breakdown and P(t) oxidation to inorganic phosphate (P(i)) in this organism. To determine if K. aerogenes ATCC 9621 could be engineered to enhance the utilization of P(n) and P(t), a multicopy plasmid, pBI05, which carried the entire phn gene cluster, was introduced into this strain. Despite the increased dosage of the phn genes, K. aerogenes ATCC 9621(pBI05) could utilize only up to 1.1-fold more P(n) and P(t) than did the control strain with the parent vector alone. These results suggested that P(i), which was generated from P(n) and P(t), might limit further utilization of these P compounds. Consequently, to convert the resulting P(i) to polyphosphate (polyP), the plasmid pKP28, which carried the K. aerogenes ppk gene (which encodes polyP kinase), was introduced into K. aerogenes ATCC 9621(pBI05). Overexpression of the ppk gene in K. aerogenes ATCC 9621(pBI05, pKP28) resulted in a 2.5-fold increase in P(t) utilization over that of the control strain. This recombinant strain also accumulated approximately sixfold more P than did the control strain when the cells were grown with MP(n) as a sole source of P.